Bone formation is a regulated and ordered developmental process that requires the biosynthetic and metabolic functions of osteoblasts. This program since its inception 18 years ago has advanced our understanding of cellular and molecular mechanisms that regulate osteoblast proliferation and differentiation, including the characterization of distinct stages of osteoblast phenotype maturation. We identified Runx2 as a transcription factor essential for osteogenic differentiation that integrates developmental signaling pathways and is a novel epigenetic regulator of cell fate determination. MicroRNAs control gene expression programs by altering both the levels and translational potential of mRNAs. One of our major discoveries in the current period is the critical role of microRNAs in controlling osteoblast lineage-commitment and maturation, as well as osteogenic signaling pathways that regulate bone mass (Li et al., Proc. Natl. Acad. Sci., 2008; Li et al, J. Biol. Chem., 2009). Our preliminary data indicate that Runx2 may regulate the expression of microRNAs that attenuate key biological pathways necessary for bone formation. Therefore, our central hypothesis is that microRNAs control commitment and differentiation of osteoblasts at key developmental transitions for regulating bone formation and that a subset of these miRs is mechanistically linked to Runx2. Consequently, we propose that developmentally expressed microRNAs can provide a novel strategy for treating skeletal disorders. In the proposed studies, we will (i) characterize how microRNAs control development of the osteoblast phenotype, (ii) analyze the function of Runx2 dependent microRNAs, and (iii) characterize skeletal phenotypes in mice defective in producing mature microRNAs in osteoblasts. The significance of our studies is the definition of mechanistic linkages among microRNAs, osteogenic signaling pathways and Runx2 in controlling osteoblast differentiation that will provide innovative insight into the molecular basis of bone formation. The principal impact of our identification of microRNAs that are rate-limiting for bone anabolic effects is the potential to develop microRNA-based pre-translational approaches as a novel dimension for clinical applications to modulate bone mass in patients.